PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A kinase

Abstract

Summary The mitotic kinase AURORA-A is essential for cell cycle progression and is considered a priority cancer target. While the catalytic activity of AURORA-A is essential for its mitotic function, recent reports indicate an additional non-catalytic function, which is difficult to target by conventional small molecules. We therefore developed a series of chemical degraders (PROTACs) by connecting a clinical kinase inhibitor of AURORA-A to E3 ligase-binding molecules (e.g. thalidomide). One degrader induced rapid, durable and highly specific degradation of AURORA-A. In addition ,we found that the degrader complex was stabilized by cooperative binding between AURORA-A and CEREBLON. Degrader-mediated AURORA-A depletion caused an S-phase defect, which is not the cell cycle effect observed upon kinase inhibition, supporting an important non-catalytic function of AURORA-A during DNA replication. AURORA-A degradation induced rampant apoptosis in cancer cell lines, and thus represents a versatile starting point for developing new therapeutics to counter AURORA-A function in cancer.

Drugging the “Undruggable” MYCN Oncogenic Transcription Factor: Overcoming Previous Obstacles to Impact Childhood Cancers

“…Small molecules have been identified that bind to AURKA and alter its conformation in a way that prevents binding to MYCN and result in rapid MYCN degradation (21). More recently, a chemical degrader approach has also been taken (22). While targeting the synthetic lethal interaction between MYCN and the kinase activity of AURKA has to-date failed to provide an efficacious and specific therapeutic, it remains possible that targeting the MYCN stabilizing function of the AURKA-MYCN complex will prove more successful.…”

Design, Synthesis and Evaluation of WD-repeat containing protein 5 (WDR5) degraders

“…Both, 8g and 17b did not induce maximal depletion of WDR5 at high concentrations, a phenomenon called the Hook-effect, resulting from less efficient ternary complex formation at excess degrader levels due to binding site competition (see Figure 3b and 3e). 35 Two negative controls, 20 resembling molecule 8g, and 21 resembling molecule 17b, with the inactive variant of the VHL ligand were synthesised to verify the effect of targeted protein degradation. Their biophysical properties can be found in the Supplementary Information.…”

“…Cycloheximide chase assay was performed as described previously. 35 MV4-11 cells were treated with 50 µg/ml CHX with or without PROTACs for different time points. The cells were harvested in RIPA buffer and probed for immunoblotting.…”

“…RT-qPCR was performed as described previously. 35 Total RNA was extracted using peqGOLD TriFast (Peqlab). cDNA synthesis was carried out and cDNA were analysed by qPCR on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) using the SYBR Green Master Mix (Thermo Fisher Scientific).…”

“…MV4-11 WDR5-HiBiT cells were used to prepare MV4-11 WDR5-HiBiT/VHL cells. HiBiT assay was performed as described previously 35. MV4-11 WDR5-HiBiT were seeded and treated with serial dilutions of compounds for 6 h or 24h.…”